Multivalent live influenza vaccine platform using recombinant adenovirus

ABSTRACT

The present invention relates to a multivalent live influenza vaccine platform using a recombinant adenovirus. The present invention is a live attenuated vaccine platform using a recombinant virus and it is easy to inoculate because it is infected with the respiratory tract like influenza virus and exhibits a vaccine action and it is a multivalent vaccine which combines two types into one and it is a highly novel vaccine that does not need to mix viruses compared to vaccines using multiple combinations of one vaccine. The present invention is the first vaccine in which a gene obtained by fusion of two influenza antigen genes into one gene is incorporated into a recombinant virus. Instead of using the entire HA gene of influenza, but using a structurally independent HA1 gene, which is about half of the total HA gene, several types of HA genes could be fused into one. When the recombinant virus was inoculated into mice by nasal inhalation, it was confirmed that it is an effective vaccine in which the vaccine effect is induced by two inoculations, and the vaccine platform of the present invention is expected to be useful for the development of a vaccine for human influenza infection.

TECHNICAL FIELD

The present invention relates to a multivalent live influenza vaccineplatform using a recombinant adenovirus and specifically, to avianinfluenza (AI virus) live vaccine using a recombinant adenovirus,wherein a vaccine candidate material capable of simultaneously defendingagainst H5 type and H7 type highly pathogenic avian influenza virus isdeveloped and prepared by linking the vaccine candidate materialharmless to the human body to an adenovirus.

BACKGROUND ART

Influenza or flu is a respiratory disease caused by infection with theinfluenza virus, and has symptoms such as chills, fever, sore throat,muscle pain, headache, and cough when infected, and in severe cases, itcauses fatal complications such as fulminant pneumonia. Human or avianinfluenza is caused by the influenza A virus, and spreads worldwide in ashort time through wild birds and travelers, causing pandemic outbreaks.Avian influenza (AI) is a type of influenza A virus, an amorphous RNAvirus with an envelope. Influenza A virus is an amorphous virus with anenvelope belonging to the Orthomyxoviridae family and is a virus whosegenes are composed of 8 segmented RNA genomes. This virus is transformedinto various types by means of mutations or recombination or mixing ofgenome fragments, causing a new type of influenza epidemic every year,and it also causes pandemic outbreak with a cycle of about 10 years.Influenza virus infects and proliferates in various host cells such ashumans, birds and pigs to produce large amounts of viral particles.Because of these characteristics, influenza viruses often have a newtype of influenza virus that has changed its infectious host throughgene exchange between species.

The types of influenza viruses are so diverse that new types of virusesappear whenever an epidemic occurs. Three types of influenza viruses areknown, A, B, and C, but most human or avian influenza epidemics arecaused by influenza A virus. Influenza A virus is classified intovarious H-type and N-type depending on the antigenicity of HA and NAproteins of envelope, and H5N1 and H7N9 types are representative highlypathogenic AI viruses. Influenza virus is very active in type conversiondue to gene mutations during the genome duplication process orrecombination or mixing of genome fragments with other types of viruses,and through such a process, a new influenza epidemic is occurring. Inaddition, since influenza viruses are infected with various animals suchas humans, birds and pigs and gene exchange between species is possible,when an AI virus is transfected to humans, it is highly likely that theAI virus will be converted to human influenza virus through type change.For example, in the case of swine flu that occurred in 2009, it is a newinfluenza virus obtained through recombination of genome fragments whileinfluenza A virus infects human, avian, and swine host cells. The novelswine-origin influenza A occurred in 2009 is a new influenza virusobtained through recombination of segmented genome when influenza Avirus infects human, avian, and swine host cells. In this regard, if thehuman infection of the Asian H5N1 and H7N9 avian influenza viruses inepidemic increases, the emergence of new human influenza viruses ispossible.

AI viruses are classified into highly pathogenic avian influenza (HPAI)and low pathogenic avian influenza (LPAI) depending on the propagationrate, mortality rate, and human infection and the H5N1 and H7N9 types,which are currently prevalent in Asia, are representative HPAIs. Becausethese HPAIs cause the death of infected individuals and contagionspreads rapidly, and thus they are classified as Class 1 infectiousdisease in domestic animals of the Domestic Animal Infectious DiseaseControl Law and high-level infection control measures are applied toprevent human infection. AI viruses can be infected to humans bycontacting the virus particles secreted by birds through the eye, noseor mouth or by inhaling the virus particles in the air. Human infectionof the AI virus has evolved into a deadly human influenza virus thatcould cause a new human influenza pandemic. HPAI virus is a deadlyinfectious disease in the poultry industry because high-levelprecautions such as poultry stamping out are applied to prevent the riskof human infection due to the rapid contagion of HPAI virus and measuressuch as chicken consumption and import/export control are taken. InKorea, avian influenza (AI) has occurred several times in Korea since2003, and in 2016, a highly pathogenic avian influenza (AI) wasprevalent and large-scale stamping-out of poultry has been carried out.H5N1 and H7N9 types are Asian HPAI viruses, and because they show a verywide host range, human infection is possible. Human infection of theseviruses has been reported repeatedly in China, and there remains apossibility of contagion to humans through poultry or migratory birds inKorea. The AI virus epidemic has been reported in Korea since 2003, andhighly pathogenic avian influenza has spread since 2016 to have a fatalimpact on the domestic poultry industry, up to now. Due to the avianinfluenza outbreak in the winter of 2016, 50 million chickens werekilled for three months, and economic losses were incurred over 1.5trillion won due to the sudden rise of egg prices, egg imports and thelike.

The development of an effective and safe poultry vaccine is the surestway to avoid concerns of human contagion as well as economic losses tofarmers. In particular, since it is difficult to predict which type ofinfluenza virus will cause the AI epidemic, it is necessary to develop amultivalent vaccine that can prevent the contagion of various types ofHPAI. In addition, as for the AI infectious disease vaccine, thedevelopment of a live vaccine is necessary, which can respondimmediately in the event of a disease, can be produced in largequantities in a short period of time and have high immune effect and canbe administered easily like respiratory vaccination. In addition, thereis a need for an efficient multivalent vaccine that can be usedprophylactically in areas where HPAI is not prevalent, and can preventvarious HPAI viruses. Currently, as a multivalent vaccine, virusesobtained by culturing several types of viruses separately and mixingthem are used. In this case, there are problems such as an imbalance inthe composition of virus types in the vaccine and bias in immuneresponse due to a specific type. In order to solve these shortcomings,to develop an AI vaccine which has high immune response and is easy toinoculate, it is urgent to develop a HPAI vaccine using a recombinantvirus that produces various types of influenza antigens from one virusparticle.

DISCLOSURE Technical Problem

An object of the present invention is a recombinant expression vectorcomprising an influenza virus H5 type hemagglutinin 1 (HA1) gene and aninfluenza virus H7 type HA1 gene, a recombinant strain transformed withthe recombinant expression vector, and a recombinant fusion protein ofinfluenza virus H5 type HA1 and H7 type HA1, which is obtained from therecombinant strain.

Another object of the present invention is to provide a method ofpreparing a recombinant adenovirus particle comprising transfecting anadenovirus with the recombinant expression vector and a recombinantadenovirus particle prepared according to the method.

Another object of the present invention is to provide a vaccinecomposition for preventing or treating influenza comprising recombinantadenovirus particles as an active ingredient, and a method of preventingor treating influenza by administering the vaccine composition toindividuals other than humans.

Technical Solution

In order to achieve the above object, the present invention provides arecombinant expression vector comprising an influenza virus H5 typehemagglutinin 1 (HA1) gene and an influenza virus H7 type HA1 gene.

Also, the present invention provides a recombinant strain transformedwith the recombinant expression vector.

In addition, the present invention provides a recombinant fusion proteinof influenza virus H5 type HA1 and H7 type HA1, which is obtained fromthe recombinant strain.

Furthermore, the present invention provides a method of preparingrecombinant adenovirus particles comprising: transfecting adenoviruswith recombinant expression vector; and culturing transfectedadenovirus.

In addition, the present invention provides recombinant adenovirusparticles prepared according to the above method.

In addition, the present invention provides a vaccine composition forpreventing or treating influenza comprising the recombinant adenovirusparticles as an active ingredient.

In addition, the present invention provides a method of preventing ortreating influenza by administering the vaccine composition toindividuals other than humans.

Advantageous Effects

The present invention relates to a multivalent live influenza vaccineplatform using a recombinant adenovirus. The present invention is a liveattenuated vaccine platform using a recombinant virus and it is easy toinoculate because it is infected with the respiratory tract likeinfluenza virus and exhibits a vaccine action. It is a multivalentvaccine which combines two types into one and it is a highly novelvaccine that does not need to mix viruses compared to vaccines usingmultiple combinations of one vaccine. The present invention is the firstvaccine in which a gene obtained by fusion of two influenza antigengenes into one gene is incorporated into a recombinant virus. Instead ofusing the entire HA gene of influenza, but using a structurallyindependent HA1 gene, which is about half of the total HA gene, severaltypes of HA genes could be fused into one. When the recombinant viruswas inoculated into mice by nasal inhalation, it was confirmed that itis an effective vaccine in which the vaccine effect is induced by twoinoculations, and the vaccine platform of the present invention isexpected to be useful for the development of a vaccine for humaninfluenza infection.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure (A) and amino acid sequence (B) of the H5type HA1 and H7 type HA1 fusion protein. H5 type HA1(1-334)-linker(335-349)-H7 type HA1(350-572) was linked to the amino terminal in theorder and Flag tag peptide is linked to 573-581, and a His tag peptideis linked to 582-587.

FIG. 2 shows the structure of the shuttle vector for adenovirusfabrication (pAd5-AI #4212) capable of expressing the H5 type HA1 and H7type HA1 fusion proteins. The fabricated fusion protein gene wasinserted between the CMV promoter of the pShuttle-CMV shuttle vector andthe SV40 polyA site in a direction capable of transcribing the gene.

FIG. 3 shows the expression results of H5 type HA1 and H7 type HA1fusion proteins. Lane 1: cell culture medium infected with controlGFP-expressing recombinant adenovirus, lane 2: cell culture mediuminfected with fusion protein-expressing recombinant adenovirus (pAd5-AI#4212 adenovirus).

FIG. 4 shows the results of anti-HA antibody production in the blood ofmice inoculated with multivalent live recombinant adenovirus vaccinethrough nasal inhalation. 1: serum of mice inhaled with GFP-expressingrecombinant adenovirus as a control, 2: serum of mice inhaled withrecombinant adenovirus expressing H5 type and H7 type HA1 fusion protein

BEST MODE

Accordingly, the present inventors used a gene in which H5 type and H7type HA genes were fused into one gene for vaccine production in orderto prepare a recombinant adenovirus vaccine against HPAI of H5 and H7types. The HA protein of the influenza virus is a protein that acts as areceptor when the virus infects host cells, and consists of an HA1portion and an HA2 portion. Even if these two parts are separated fromeach other, they maintain an independent structure without anymodification of their structure in the HA protein, and the HA1 partalone can bind to the host cell receptor. Based on these points, afusion gene was prepared by combining two reduced H5 type and H7 typeHA1 genes into one gene. By linking two H5 and H7 HA1 genes into onegene, the overall gene size is not excessively large, so a recombinantadenovirus containing the fused gene can be produced and thisrecombinant virus can be used as a multivalent live influenza vaccine tocomplete the present invention.

The present invention provides a recombinant expression vectorcomprising an influenza virus H5 type hemagglutinin 1 (HA1) gene and aninfluenza virus H7 type HA1 gene.

Preferably, the recombinant expression vector may comprise a ribosomebinding site, an influenza virus H5 type HA1 gene, a linker, aninfluenza virus H7 type HA1 gene and a tag gene in order, but it is notlimited thereto.

More preferably, the influenza virus H5 type HA1 gene may be representedby SEQ ID NO: 2, and the influenza virus H7 type HA1 gene may berepresented by SEQ ID NO: 3, but they are not limited thereto.

More preferably, the ribosome binding site may be RBS/Kozak(aaggaggccgccacc) to help the protein decoding, and the linker may beglycine/serine linker peptide, as a role of serving to link two genesfor expression, and, and the tag gene may be a Flag tag (DYKDDDDKG) andHis tag (HHHHHH) genes, as a role of serving to facilitate purificationof the protein for expression, but they are not limited thereto.

More preferably, the influenza virus may be an influenza A virus, but itis not limited thereto.

In the present invention, “hemagglutinin (HA)” is one of the surfaceglycoproteins of influenza virus and mediates viral-cell membrane fusionduring adhesion of the virus to host cells and cell penetration of thevirus. It is a surface antigen protein of influenza virus thatdetermines antigen specificity.

In the present invention, “vector” refers to a DNA molecule thatreplicates itself and is used to carry a clone gene (or other fragmentof clone DNA).

In the present invention, “expression vector” refers to a recombinantDNA molecule comprising a target coding sequence and an appropriatenucleic acid sequence essential for expressing a coding sequenceoperably linked in a specific host organism. The expression vector maypreferably contain at least one selectable marker. The marker istypically a nucleic acid sequence having a property that can be selectedby a chemical method, and includes all genes capable of distinguishing atransformed cell from a non-transformed cell. Examples includeantibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin(G418), Bleomycin, Hygromycin, and Chloramphenicol, but it is limitedthereto, and can be appropriately selected by a person skilled in theart.

In order to express the DNA sequence of the present invention, any of awide variety of expression regulatory sequences can be used in thevector. Examples of useful expression regulatory sequences may include,for example, early and late promoters of SV40 or adenovirus, promotersand enhancers of CMV, LTR of retroviruses, lac system, trp system, TACor TRC system, T3 and T7 promoters, main operator and promoter region ofphage lambda, the regulatory region of the fd code protein, the promoterfor 3-phosphoglycerate kinase or other glycolase, the promoters of thephosphatase such as Pho5, alpha-crossing system promoters of yeast andconstructs and other induced sequences known to regulate the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof.

In addition, the present invention provides a recombinant straintransformed with the recombinant expression vector. Preferably, therecombinant strain may be an adenovirus, but it is not limited thereto.

In addition, the present invention provides a recombinant fusion proteinof influenza virus H5 type HA1 and H7 type HA1, which is obtained fromthe recombinant strain.

Preferably, the recombinant fusion protein may be represented by SEQ IDNO: 1, but it is not limited thereto.

In the present invention, “fusion protein” means that one or morepolypeptides are bound to a single strand of polypeptide through apeptide bond, and it refers to a fusion protein prepared by fusion ofone or more genes including a target gene by a recombinant method andtranslation into a single polypeptide. In the present invention, thefusion protein may include an amino acid cleavage sequence (chemicalmaterials or enzyme specific cleavage sequence) which can be cleaved byspecifically recognizing by a chemical substance or enzyme betweenproteins or peptide sequences of interest which are fused to each other.

In addition, the present invention provides a method of preparingrecombinant adenovirus particles comprising: transfecting adenoviruswith recombinant expression vector; and culturing transfectedadenovirus. Preferably, the recombinant adenovirus particles may expressa recombinant fusion protein of influenza virus H5 type HA1 and H7 typeHA1. More preferably, the recombinant fusion protein may be representedby SEQ ID NO: 1, but it is not limited thereto.

Furthermore, the present invention provides recombinant adenovirusparticles prepared according to the above method.

In addition, the present invention provides a vaccine composition forpreventing or treating influenza comprising the recombinant adenovirusparticles as an active ingredient.

Host animals capable of causing an immune response by the vaccine of thepresent invention may include humans, dogs, cats, pigs, horses,chickens, ducks, turkeys, ferrets, etc., and preferably, the influenzamay be avian influenza, swine influenza or human influenza, but it isnot limited thereto.

The vaccine of the present invention may be an attenuated live vaccineor killed vaccine, a subunit vaccine, a synthetic vaccine or a geneticengineering vaccine, but live vaccines that induce an effective immuneresponse are preferred.

In the present invention, the term “live vaccine” or “live attenuatedvaccine” refers to a vaccine containing a live viral active ingredient.In addition, “attenuation” means that the toxicity of a living pathogenis artificially weakened, and it does not cause disease in the body bymutating genes involved in the essential metabolism of pathogens, butonly stimulates the immune system to induce immunity. Viral attenuationcan be achieved by ultraviolet (UV) irradiation, chemical treatment, orhigh-order continuous subculture in vitro. Attenuation can also beachieved by making clear genetic changes, for example by specificdeletions of viral sequences known to provide toxicity or by insertionof sequences into the viral genome.

In addition, the vaccine of the present invention may further contain atleast one selected from the group consisting of a solvent, an adjuvantand an excipient. Examples of the solvent include physiological salineor distilled water, and examples of immune enhancing agents includeFreund's incomplete or complete adjuvant, aluminum hydroxide gel andvegetable and mineral oils, and excipients include aluminum phosphate,aluminum hydroxide, or aluminum potassium sulfate, but they are notlimited thereto, and may further include a material used in thepreparation of a vaccine, well known to those skilled in the art.

In addition, the vaccine of the present invention may be prepared in anoral or parenteral formulation, and may be administered by intradermal,intramuscular, intraperitoneal, nasal or epidural routes.

Furthermore, the present invention provides a method of preventing ortreating influenza by administering the vaccine composition toindividuals other than humans.

In the present invention, “individual” means all animals, includinghumans, who have already been infected with or can be infected withinfluenza virus. The disease can be efficiently prevented and treated byadministering the vaccine composition of the present invention to anindividual. For example, the composition of the present invention cantreat humans infected with various influenza virus subtypes or variantsof influenza virus. In addition, the composition of the presentinvention can treat chickens or pigs infected with various influenzavirus subtypes or variants of avian influenza. The composition of thepresent invention can be administered in combination with theconventional therapeutic agent for influenza virus infection disease.

In the present invention, “prevention” means any action of inhibiting ordelaying the onset of influenza virus infection by administration of avaccine composition. In addition, “treatment” refers to any action inwhich symptoms caused by influenza virus infection are improved orbeneficially changed by administration of a vaccine composition.

The vaccine composition of the present invention is administered in apharmaceutically effective amount. The term “pharmaceutically effectiveamount” means an amount sufficient to treat a disease with a reasonablebenefit/risk ratio applicable to medical treatment, and the effectivedose level may be determined by factors including the type and severityof the individual, age, sex, type of infected virus, drug activity,sensitivity to drugs, time of administration, route of administration,rate of excretion, treatment period, drugs used concurrently, and otherfactors well known in the medical field. The vaccine composition of thepresent invention may be administered as an individual therapeutic agentor administered in combination with other therapeutic agents, and may beadministered sequentially or simultaneously with a conventionaltherapeutic agent. Also, it can be administered single or multiple. Itis important to administer an amount capable of obtaining the maximumeffect in a minimum amount without side effects in consideration of allthe above factors, and can be easily determined by a person skilled inthe art.

Hereinafter, the present invention will be described in more detailthrough examples. These examples are only intended to illustrate thepresent invention in more detail, and it will be apparent to thoseskilled in the art that the scope of the present invention is notlimited by these examples according to the gist of the presentinvention.

<Example 1> Preparation of H5 Type and H7 Type HA1 Fusion Protein (H5/H7Fusion Protein) Genes

In order to secure the nucleic sequence of the H5 type HA1 gene, inamino acid sequences of the HA protein of the influenza A virus(A/goose/Guangdong/1/1996(H5N1)) isolated from goose in Guangdong, Chinain 1996 in Gene Bank Database (NC_007362.1), the amino acid sequencesfrom amino terminals 1 to 346 corresponding to the HA1 region wereselected. This amino acid sequence contains the leader peptide portionof the HA protein and the protein coding region of the portioncorresponding to the HA1 region. This region contains thereceptor-binding site and esterase part of the HA protein, but excludesfusion peptide, HA2 part, and transmembrane region. To secure thenucleic sequence of the H7 type HA1 gene, in amino acid sequence of theHA protein of the influenza A virus (A/Chicken/Italy/1067/99(H7N1))isolated from Italian chickens in 1999 in the Gene Bank Database(AJ584647.1) amino acid sequences from amino terminals 17 to 339corresponding to the HA1 region were selected. This region contains thereceptor-binding site and esterase part of the HA protein, but excludesleader peptide part, fusion peptide, HA2 part, and transmembrane region.These two HA1 proteins were linked in the order of H5 typeHA1(1-334)-linker(335-349)-H7 type HA1(350-572) with glycine/serinelinker peptide to express one protein. In order that the linked peptidesare effectively decoded in eukaryotic cells or E. coli, and can be usedfor identification and separation of proteins expressed on the carboxyterminal side of the linked peptide amino acids, peptides that act asflag tags (DYKDDDDKG) and His tags (HHHHHH) are linked to design aprotein composed of a total of 587 amino acids (FIG. 1 and SEQ ID NO:1).

In order to prepare a recombinant virus expressing the protein asproduced in animal cells, the codon nucleotide sequence corresponding tothe amino acid sequence of this protein was determined by optimizing thecodon in a form that can be read best in animal cells. Finally, a geneexpressing the fusion protein was synthesized by adding the RBS/Kozaknucleotide sequence, which is a ribosome binding site that helps proteindecoding in front of the entire nucleotide sequence (RBS KOZAK/HA1(H5)(1-346; SEQ ID NO: 2)/GS linker(15)/HA1(H7)(17-339; SEQ ID NO:3)/FLAG/His6tga)(SEQ ID NO: 4). Gene synthesis was performed using CosmoGenetech's gene synthesis service.

<Example 2> Expression of H5 Type and H7 Type HA1 Fusion Protein

In order to confirm the production of the fusion protein from the geneproduced as described above, the synthesized gene was inserted into aeukaryotic expression vector to produce a recombinant adenovirusparticle containing the expression vector. To produce fusion proteingene, firstly, a pAd5-AI #4212 clone inserted in a direction in whichthe gene can be transcribed between the CMV promoter of the pShuttle-CMVshuttle vector and the SV40 polyA site, required for the production ofrecombinant adenovirus was obtained (FIG. 2 ).

The pAd5-AI #4212 DNA was investigated by Genuine Tech Co., Ltd. toconfirm that the fusion protein can be produced in animal cells usingrecombinant adenovirus particles containing this plasmid. To this end,2×10⁵ HEK293A cells were cultured overnight, and then 2×10⁹ pfu ofpAd5-AI #4212 recombinant adenovirus particles were mixed in the culturesolution so that the MOU became 10,000, and the cells were infected, andthen incubated for 24 hours in a medium containing fetal bovine serum.Thereafter, the medium was replaced with a medium without fetal bovineserum, and then cultured for 24 hours, and then the culture medium wascollected. In this way, the culture medium of the infected cells wascollected three times to recover the fusion protein. The culture mediumcontaining the collected fusion protein was concentrated 20-fold, andthen the production of fusion protein was investigated by a westernblotting method using a Flag tag. As a control, a recombinant adenovirusexpressing green fluorescent protein (GFP) was used instead of thefusion protein. The concentrated culture was separated by SDS-PAGE, andthe protein was transferred to the NC Membrane, reacted with a mouseanti-Flag antibody, and then reacted with a secondary antibody,Rat-anti-mouse IgG1-HRP (eBioscience). After the reaction was completed,the reaction was washed with PBS and then reacted with WEST-Queen™ kit(iNtRON), and the presence or absence of the protein reacting with theFlag tag antibody and its size was measured using a fluorescence imageanalyzer (Fusion-SL4) (FIG. 3 ). As a result, proteins that react withthe Flag tag antibody were not identified in the cell culture mediuminfected with GFP-expressing recombinant adenovirus used as a control,but in the cell culture medium infected with the recombinant adenovirusexpressing the fusion protein (pAd5-AI #4212 adenovirus), it was foundthat 50 kDa and 100 kDa proteins reacted with the Flag tag antibody.These results show that the expression construct produces a successfullyfused protein from the produced H5 type HA1 and H7 type HA1 fusionproteins.

<Example 3> Effect of Inducing Antibody Production by RecombinantAdenovirus Expressing H5 and H7 Types HA1 Fusion Proteins in Mice

Four five-week-old female BALB/c mice were inoculated with pAd5-AI #4212recombinant adenovirus particles expressing H5 type and H7 type HA1fusion protein by intranasal instillation. As a control, 4 mice wereinoculated with a recombinant adenovirus expressing green fluorescentprotein (GFP) instead of the fusion protein by intranasal inhalation.For intranasal inhalation, 5×10⁸ pfu recombinant adenovirus particleswere diluted in 30 μl of a phosphate buffer solution and 5 μl of eachwas alternately inhaled using both nose holes. After the completion ofthe first inoculation and breeding for 3 weeks, and then the virus wasinoculated again in the same manner. One week after the inoculation,blood was collected from the eyes of mice to prepare antisera.

The presence or absence of antibody against the fusion protein in theserum obtained by inoculating a recombinant adenovirus expressing thefusion protein twice by intranasal inhalation was confirmed usingEnzyme-linked ImmunoSorbent Assay (ELISA). First, the H5 type and H7type HA1 fusion protein culture solution obtained from cell culture wereadded to the ELISA plate and reacted overnight at 4° C. to coat thefusion protein. The next day, the serum of the mice inoculated with thefusion protein recombinant adenovirus was diluted 500 times and reactedfor 2 hours at room temperature, and then reacted with theHRP-conjugated anti-mouse IgG/M secondary antibody (Merck MilliporeAP130P) and antigen-antibody reaction was measured at 450 nm with anELISA leader (ThermoFisher MULTISKAN GO). As a result, it was confirmedthat antibodies reacting with the fusion protein were formed in theserum of mice inhaled with the recombinant adenovirus expressing the H5type and H7 type HA1 fusion proteins (FIG. 4 ).

These results show that the recombinant adenovirus expressing the H5type and H7 type HA1 fusion proteins was infected in the respiratorymucosa like a normal adenovirus, and the antibody response was inducedin the mouse immune system in a similar manner as when infected with theinfluenza virus. In other words, it is suggested that the recombinantadenovirus having the HA fusion protein gene of the influenza virus canbe effectively infected in the respiratory tract of an animal to be usedas a live vaccine that effectively induces an immune response to theinserted influenza protein. Influenza vaccine using this type of fusionprotein is a multivalent vaccine that simultaneously provides two ormore influenza antigens to the immune system and it is a new preparationmethod of influenza vaccine that has not been attempted at all so far.These vaccines offer several advantages over multivalent vaccines usinga mixture of several types of influenza viruses.

The multivalent influenza live vaccine platform in which multipleantigens are bound to one recombinant viral particle as the presentinvention does not require the process of producing and mixing eachmonovalent vaccine virus, so it is expected to be able to quicklyrespond to avian influenza transmission. Also, because recombinantadenovirus is a live virus, it can induce a strong immune responseagainst influenza virus as in AI virus infection and it is a liveattenuated vaccine, it can be easily administered by respiratoryinhalation like influenza virus infection, and mass vaccination ispossible, and it is a vaccine manufacturing method suitable for poultryvaccination. In addition, since recombinant adenovirus is a relativelyharmless virus and does not use highly pathogenic influenza virus forvaccine production, the risk of biosafety in the manufacturing processis low, and it will further contribute to shorten the production periodand to reduce the production cost.

While the present invention has been particularly described withreference to specific embodiments thereof, it is apparent that thisspecific description is only a preferred embodiment and that the scopeof the present invention is not limited thereby to those skilled in theart. That is, the practical scope of the present invention is defined bythe appended claims and their equivalents.

The invention claimed is:
 1. A recombinant fusion protein of influenza Avirus H5 type hemagglutinin 1 (HA1) and H7 type HAL which is obtainedfrom a recombinant adenovirus strain transfected with a recombinantexpression vector comprising an influenza A virus H5 type hemagglutinin1 (HA1) gene and an influenza A virus H7 type HA1 gene.
 2. Therecombinant fusion protein of claim 1, wherein the recombinantexpression vector comprises a ribosome binding site, an influenza Avirus H5 type HA1 gene, a linker, an influenza A virus H7 type HA1 geneand a tag gene in order.
 3. The recombinant fusion protein of claim 1,wherein the influenza A virus H5 type HA1 gene is represented by SEQ IDNO: 2, and the influenza A virus H7 type HA1 gene is represented by SEQID NO:
 3. 4. The recombinant fusion protein of claim 1, wherein therecombinant fusion protein is represented by SEQ ID NO:
 1. 5. A methodof preparing recombinant adenovirus particles comprising: transfectingadenovirus with a recombinant expression vector comprising an influenzavirus H5 type HA1 gene and an influenza virus H7 type HA1 gene; andculturing transfected adenovirus.
 6. The method of preparing recombinantadenovirus particles of claim 5, wherein the recombinant adenovirusparticles express influenza virus H5 type HA1 and H7 type HA1recombinant fusion proteins.
 7. The method of preparing recombinantadenovirus particles of claim 6, wherein the recombinant fusion proteinis represented by SEQ ID NO:
 1. 8. A method of treating influenza,comprising: providing a vaccine composition comprising the recombinantfusion protein of claim 1 as an active ingredient; and administering thevaccine composition to a subject.
 9. The method of treating influenza ofclaim 8, wherein the influenza is avian influenza, swine influenza orhuman influenza.